The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Maarten Merkx - One of the best experts on this subject based on the ideXlab platform.
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tuning the flexibility of glycine serine Linkers to allow rational design of multidomain proteins
Biochemistry, 2017Co-Authors: Martijn M Van Rosmalen, Mike Krom, Maarten MerkxAbstract:Flexible polypeptide Linkers composed of glycine and serine are important components of engineered multidomain proteins. We have previously shown that the conformational properties of Gly-Gly-Ser repeat Linkers can be quantitatively understood by comparing experimentally determined Forster resonance energy transfer (FRET) efficiencies of ECFP-linker-EYFP proteins to theoretical FRET efficiencies calculated using wormlike chain and Gaussian chain models. Here we extend this analysis to include Linkers with different glycine contents. We determined the FRET efficiencies of ECFP-linker-EYFP proteins with Linkers ranging in length from 25 to 73 amino acids and with glycine contents of 33.3% (GSSGSS), 16.7% (GSSSSSS), and 0% (SSSSSSS). The FRET efficiency decreased with an increasing linker length and was overall lower for Linkers with less glycine. Modeling the Linkers using the WLC model revealed that the experimentally observed FRET efficiencies were consistent with persistence lengths of 4.5, 4.8, and 6.2 ...
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variation of linker length in ratiometric fluorescent sensor proteins allows rational tuning of zn ii affinity in the picomolar to femtomolar range
Journal of the American Chemical Society, 2007Co-Authors: E M W M Van Dongen, Toon H Evers, E W Meijer, L M Dekkers, Leo W J Klomp, Maarten MerkxAbstract:Ratiometric fluorescent sensor proteins with a very high and tunable affinity for Zn(II) were created by connecting two fluorescently labeled metal binding domains, CFP−Atox1 and WD4−YFP, using a series of flexible peptide Linkers. A simple random-coil model describing the conformational distribution of the linker allowed a quantitative understanding of the effect of the linker length on both the change in emission ratio and the Zn(II) affinity.
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quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide Linkers
Biochemistry, 2006Co-Authors: Toon H Evers, Elisabeth M W M Van Dongen, Alex C Faesen, E W Meijer, Maarten MerkxAbstract:The fusion of different protein domains via peptide Linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide Linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide Linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP−linker−EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistenc...
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quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide Linkers
Biochemistry, 2006Co-Authors: Toon H Evers, Alex C Faesen, E W Meijer, Elisabeth M W M Van Dongen, Maarten MerkxAbstract:The fusion of different protein domains via peptide Linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide Linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide Linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser Linkers are discussed.
Mingguang Feng - One of the best experts on this subject based on the ideXlab platform.
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bifunctional enhancement of a beta glucanase xylanase fusion enzyme by optimization of peptide Linkers
Applied Microbiology and Biotechnology, 2008Co-Authors: Mingguang FengAbstract:The flexible peptides (GGGGS)n (n < or = 3), the alpha-helical peptides (EAAAK)n (n < or = 3) and two other peptides were used as Linkers to construct bifunctional fusions of beta-glucanase (Glu) and xylanase (Xyl) for improved catalytic efficiencies of both moieties. Eight Glu-Xyl fusion enzymes constructed with different Linkers were all expressed as the proteins of ca. 46 kDa in Escherichia coli BL21 and displayed the activities of both beta-glucanase and xylanase. Compared to all the characterized fusions with the parental enzymes, the catalytic efficiencies of the Glu and Xyl moieties were equivalent to 304-426% and 82-143% of the parental ones, respectively. The peptide linker (GGGGS)(2) resulted in the best fusion, whose catalytic efficiency had a net increase of 326% for the Glu and of 43% for the Xyl. The two moieties of a fusion with the linker (EAAAK)(3) also showed net increases of 262 and 31% in catalytic efficiency. Our results highlight, for the first time, the enhanced bifunctional activities of the Glu-Xyl fusion enzyme by optimizing the peptide Linkers to separate the two moieties at a reasonable distance for beneficial interaction.
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bifunctional enhancement of a beta glucanase xylanase fusion enzyme by optimization of peptide Linkers
Applied Microbiology and Biotechnology, 2008Co-Authors: Mingguang FengAbstract:The flexible peptides (GGGGS)n (n ≤ 3), the α-helical peptides (EAAAK)n (n ≤ 3) and two other peptides were used as Linkers to construct bifunctional fusions of β-glucanase (Glu) and xylanase (Xyl) for improved catalytic efficiencies of both moieties. Eight Glu-Xyl fusion enzymes constructed with different Linkers were all expressed as the proteins of ca. 46 kDa in Escherichia coli BL21 and displayed the activities of both β-glucanase and xylanase. Compared to all the characterized fusions with the parental enzymes, the catalytic efficiencies of the Glu and Xyl moieties were equivalent to 304–426% and 82–143% of the parental ones, respectively. The peptide linker (GGGGS)2 resulted in the best fusion, whose catalytic efficiency had a net increase of 326% for the Glu and of 43% for the Xyl. The two moieties of a fusion with the linker (EAAAK)3 also showed net increases of 262 and 31% in catalytic efficiency. Our results highlight, for the first time, the enhanced bifunctional activities of the Glu-Xyl fusion enzyme by optimizing the peptide Linkers to separate the two moieties at a reasonable distance for beneficial interaction.
Toon H Evers - One of the best experts on this subject based on the ideXlab platform.
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variation of linker length in ratiometric fluorescent sensor proteins allows rational tuning of zn ii affinity in the picomolar to femtomolar range
Journal of the American Chemical Society, 2007Co-Authors: E M W M Van Dongen, Toon H Evers, E W Meijer, L M Dekkers, Leo W J Klomp, Maarten MerkxAbstract:Ratiometric fluorescent sensor proteins with a very high and tunable affinity for Zn(II) were created by connecting two fluorescently labeled metal binding domains, CFP−Atox1 and WD4−YFP, using a series of flexible peptide Linkers. A simple random-coil model describing the conformational distribution of the linker allowed a quantitative understanding of the effect of the linker length on both the change in emission ratio and the Zn(II) affinity.
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quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide Linkers
Biochemistry, 2006Co-Authors: Toon H Evers, Elisabeth M W M Van Dongen, Alex C Faesen, E W Meijer, Maarten MerkxAbstract:The fusion of different protein domains via peptide Linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide Linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide Linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP−linker−EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistenc...
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quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide Linkers
Biochemistry, 2006Co-Authors: Toon H Evers, Alex C Faesen, E W Meijer, Elisabeth M W M Van Dongen, Maarten MerkxAbstract:The fusion of different protein domains via peptide Linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide Linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide Linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser Linkers are discussed.
Philip N. Bartlett - One of the best experts on this subject based on the ideXlab platform.
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covalent modification of glassy carbon surface with organic redox probes through diamine Linkers using electrochemical and solid phase synthesis methodologies
Journal of Materials Chemistry, 2008Co-Authors: Mohamed A Ghanem, Jeanmathieu Chretien, Aleksandra Pinczewska, Jeremy D. Kilburn, Philip N. BartlettAbstract:Various mono-Boc-protected diamines have been covalently grafted to glassy carbon electrodes by electrochemical oxidation of the free amine. After deprotection of the Boc group, anthraquinone and nitrobenzene probes were coupled to the Linkers using solid-phase coupling reactions. X-Ray photoelectron spectroscopy and cyclic voltammetry were used to monitor the coupling efficiency, effect of linker length on the surface coverage and electron transfer between the attached redox probes and electrode. The anthraquinone surface coverage was found to decrease as the chain length of alkyl diamine linker increased and the electron transfer kinetics were found to be faster for the lower coverages and the longer, more flexible Linkers. In the case of nitrobenzene, there was only a slightly change in coverage with increasing linker length. This electrochemical attachment of protected diamine Linkers followed by solid-phase coupling provides a very versatile methodology for attaching a wide range of molecular architectures onto glassy carbon surfaces.
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covalent tethering of organic functionality to the surface of glassy carbon electrodes by using electrochemical and solid phase synthesis methodologies
Chemistry: A European Journal, 2008Co-Authors: Jeanmathieu Chretien, Mohamed A Ghanem, Philip N. Bartlett, Jeremy D. KilburnAbstract:Organic Linkers such as (N-Boc-aminomethyl)phenyl (BocNHCH2C6H4) and N-Boc-ethylenediamine (Boc-EDA) have been covalently tethered onto a glassy carbon surface by employing electrochemical reduction of BocNHCH2C6H4 diazonium salt or oxidation of Boc-EDA. After removal of the Boc group, anthraquinone as a redox model was attached to the linker by a solid-phase coupling reaction. Grafting of anthraquinone to electrodes bearing a second spacer such as 4-(N-Boc-aminomethyl)benzoic acid or N-Boc--alanine was also performed by following this methodology. The surface coverage, stability and electron transfer to/from the tethered anthraquinone redox group through the Linkers were investigated by cyclic voltammetry. The effects of pH and scan rate were studied, and the electron-transfer coefficient and rate constant were determined by using Laviron's equation for the different types of linker. The combination of electrochemical attachment of protected Linkers and subsequent modifications under the conditions of solid-phase synthesis provides a very versatile methodology for tailoring a wide range of organic functional arrangements on a glassy carbon surface.
Jeremy D. Kilburn - One of the best experts on this subject based on the ideXlab platform.
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covalent modification of glassy carbon surface with organic redox probes through diamine Linkers using electrochemical and solid phase synthesis methodologies
Journal of Materials Chemistry, 2008Co-Authors: Mohamed A Ghanem, Jeanmathieu Chretien, Aleksandra Pinczewska, Jeremy D. Kilburn, Philip N. BartlettAbstract:Various mono-Boc-protected diamines have been covalently grafted to glassy carbon electrodes by electrochemical oxidation of the free amine. After deprotection of the Boc group, anthraquinone and nitrobenzene probes were coupled to the Linkers using solid-phase coupling reactions. X-Ray photoelectron spectroscopy and cyclic voltammetry were used to monitor the coupling efficiency, effect of linker length on the surface coverage and electron transfer between the attached redox probes and electrode. The anthraquinone surface coverage was found to decrease as the chain length of alkyl diamine linker increased and the electron transfer kinetics were found to be faster for the lower coverages and the longer, more flexible Linkers. In the case of nitrobenzene, there was only a slightly change in coverage with increasing linker length. This electrochemical attachment of protected diamine Linkers followed by solid-phase coupling provides a very versatile methodology for attaching a wide range of molecular architectures onto glassy carbon surfaces.
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covalent tethering of organic functionality to the surface of glassy carbon electrodes by using electrochemical and solid phase synthesis methodologies
Chemistry: A European Journal, 2008Co-Authors: Jeanmathieu Chretien, Mohamed A Ghanem, Philip N. Bartlett, Jeremy D. KilburnAbstract:Organic Linkers such as (N-Boc-aminomethyl)phenyl (BocNHCH2C6H4) and N-Boc-ethylenediamine (Boc-EDA) have been covalently tethered onto a glassy carbon surface by employing electrochemical reduction of BocNHCH2C6H4 diazonium salt or oxidation of Boc-EDA. After removal of the Boc group, anthraquinone as a redox model was attached to the linker by a solid-phase coupling reaction. Grafting of anthraquinone to electrodes bearing a second spacer such as 4-(N-Boc-aminomethyl)benzoic acid or N-Boc--alanine was also performed by following this methodology. The surface coverage, stability and electron transfer to/from the tethered anthraquinone redox group through the Linkers were investigated by cyclic voltammetry. The effects of pH and scan rate were studied, and the electron-transfer coefficient and rate constant were determined by using Laviron's equation for the different types of linker. The combination of electrochemical attachment of protected Linkers and subsequent modifications under the conditions of solid-phase synthesis provides a very versatile methodology for tailoring a wide range of organic functional arrangements on a glassy carbon surface.
